The primary goal of these studies is to analyze kinetic and biochemical aspects of diacylglycerol metabolism in cultured fibroblasts under conditions which differentially stimulate the generation of this lipid. Recently, evidence has been presented indicating that thrombin activates at least two coupling mechanisms for the stimulation of lipid metabolism in cultured fibroblasts: (1) R1 which (a) responds to alpha-thrombin but not gamma-thrombin, (b) is inactivated by chymotrypsin, and (c) stimulates both the hydrolysis of polphosphoinositides and release of arachidonic acid; and (2) R2 which (a) responds to both alpha-thrombin and gamma-thrombin, (b) is chymotrypsin-insensitive and (c) does not stimulate the hydrolysis of polyphosphoinositides and release of arachidonic acid. Activation of either R1 and R2 results in the elevation of cellular diacylglycerol levels. PDGF activates R1-like activities while EGF activates R2-like activities. The stimulated metabolism of diacylglycerol will be analyzed under conditions which differentiate between R1 and R2. Recently, using a highly sensitive assay capable of accurately measuring mass amounts of diacylglycerol, we have examined the kinetics of diacylglycerol production stimulated by alpha-thrombin and EGF. We have begun to examine the dose-dependence and chymotrypsin sensitivity of alpha-thrombin-stimulated diacylglycerol production. We now plan to extend these studies and to include an evaluation of gamma-thrombi and PDGF stimulated diacylglycerol production and to identify the molecular species of the diacylglycerols stimulated by these molecules. While examining the stimulation by EGF or PDGF, experiments will be conducted to examine the relationship between receptor occupancy and the stimulation of diacylglycerol. The role of the high affinity receptor for thrombin will also be examined to determine its potential role in the activation of R1 and R2. In summary, experiments have been designed to qualitatively and quantitatively analyze the diacylglycerols stimulated under conditions which differentiate between R1 and R2, and to examine cellular phosphoinositides as a potential source of these diacylglycerols. These analyses will provide valuable information concerning the role of stimulated diacylglycerol in modulating short term and long term responses by hormones and growth factors.